(1) To develop animal disease (end host) and persistence (reservoir host) models: We have developed a nonhuman primate model for the Bangladesh genotype of Nipah virus in African green monkeys. The availability of this model is of great importance since the vast majority of human cases of Nipah virus since 1999 were caused by viruses of the Bangladesh genotype. The results suggest that the pathogenesis of this genotype is like that of the Malaysia genotype, but with accelerated disease progression in the end stage. (de Wit et al., unpublished studies ongoing) Disease severity of Middle East Respiratory Syndrome (MERS)has been linked to patient health status, as people with chronic diseases or an immunocompromised status fare worse, although the mechanisms of disease have yet to be elucidated. We used the rhesus macaque model of mild MERS to investigate whether the immune response plays a role in the pathogenicity in relation to MERS-CoV shedding. Immunosuppressed macaques supported significantly higher levels of MERS-CoV replication in respiratory tissues and shed more virus, and virus disseminated to tissues outside of the respiratory tract, whereas viral RNA was confined to respiratory tissues in non-immunosuppressed animals. Despite increased viral replication, pathology in the lungs was significantly lower in immunosuppressed animals. The observation that the virus was less pathogenic in these animals suggests that disease has an immunopathogenic component and shows that inflammatory responses elicited by the virus contribute to disease. (Prescott et al., Front Immunol 2018) We tested the suitability of the domestic pig as a model for MERS-CoV infection. Inoculation did not cause disease, but a low level of virus replication, shedding, and seroconversion were observed. Pigs do not recapitulate human MERS-CoV and are unlikely to constitute a reservoir in nature. (de Wit et al., Emerg Infect Dis 2017) (2) To identify and characterize determinants of viral pathogenicity to develop antivirals: We performed a time-course study in ferrets inoculated intranasally with 1918 H1N1 influenza virus, with special emphasis on the involvement of extrarespiratory tissues. Respiratory and extrarespiratory tissues were collected after inoculation for virologic, histological, and immunological analysis. Infectious virus was detected at high titers in respiratory tissues and, at lower titers in most extrarespiratory tissues. Evidence for active virus replication, as indicated by the detection of nucleoprotein by immunohistochemistry, was observed in the respiratory tract, peripheral and central nervous system, and liver. Proinflammatory cytokines were up-regulated in respiratory tissues, olfactory bulb, spinal cord, liver, heart, and pancreas. 1918 H1N1 virus spread to and induced cytokine responses in tissues outside the respiratory tract, which likely contributed to the severity of infection. Moreover, our data support the suggested link between 1918 H1N1 infection and central nervous system disease. (de Wit et al. J Infect Dis 2018) Influenza A virus (IAV) infection can be associated with secondary bacterial coinfection, and it has long been posited that the ability of IAV to alter normal neutrophil function predisposes individuals to secondary bacterial infections. To better understand this phenomenon, we evaluated the interaction of pandemic or seasonal H1N1 IAV with human neutrophils isolated from healthy persons. These viruses were ingested by human neutrophils and elicited changes in neutrophil gene expression that are consistent with an interferon-mediated immune response. The viability of neutrophils following coculture with either pandemic or seasonal H1N1 IAV was similar for up to 18 h of culture. Notably, neutrophil exposure to seasonal (but not pandemic) IAV primed these leukocytes for enhanced functions, including production of reactive oxygen species and bactericidal activity. Taken together, our results are at variance with the universal idea that IAV impairs neutrophil function directly to predispose individuals to secondary bacterial infections. Rather, we suggest that some strains of IAV prime neutrophils for enhanced bacterial clearance. (Malachowa et al. mSphere 2018) (3) To identify and characterize host responses to viral infection to develop therapeutics: In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV. The post-exposure efficacy of nebulized griffithsin in the rhesus macaque model showed moderate reduction of viral load but did not significantly reduce disease signs. We have now shown that pre-exposure treatment reduces clinical signs of disease and viral titers in target organs. (Falzarano et al., manuscript in preparation) We have also tested efficacy of three monoclonal antibodies (mAb) as a treatment for MERS-CoV infection in the common marmoset. These mAb had shown efficacy in mouse models of MERS-CoV infection. Unfortunately, none of the mABs showed significant reduction in disease burden and viral lung load in the nonhuman primate model suggesting that treatment with mABs may likely not very efficacious. Confirmatory studies and treatment with mAB cocktails are either ongoing or planned. (van Doremalen et al Antiviral Res 2017; de Wit et al. Antiviral Res 2018) We have tested the efficacy of the antiviral compound GS-5734 against MERS-CoV in the rhesus macaque model. Pre-exposure treatment resulted in reduction of disease burden and viral lung loads. In contrast, post-exposure treatment with GS-5734 showed only minor effects. (de Wit et al., manuscript in preparation) We also tested the efficacy of GS-5734 against Nipah virus (Bangladesh genotype) in African green monkeys. Animals were inoculated with a lethal dose of Nipah virus and a once-daily intravenous GS-5734 treatment was initiated one day later. Mild respiratory signs were observed in 2 of 4 treated animals, whereas all control animals developed severe respiratory disease signs. In contrast to control animals, which all succumbed to the infection, all GS-5734-treated animals survived the lethal challenge, indicating that GS-5734 is a promising antiviral treatment for Nipah virus infection. (Lo et al. submitted) (4) To develop protective vaccines: For MERS, we continued testing a promising DNA vaccine platform encoding a codon-optimized consensus spike protein. This vaccine induced potent cellular immunity and antigen specific neutralizing antibodies in three animal species using a prime/boost/boost approach. Vaccinated macaques were protected against MERS-CoV challenge and did not show any clinical or radiographic signs of pneumonia. Recently, we were successful in shortening the vaccination strategy for potential application of this vaccination approach in emergency situations to prevent MERS-CoV infection. (Falzarano et al, manuscript in preparation) To generate a vaccine against Nipah virus infection, we used the VSV platform to express single Nipah virus glycoproteins (G or F) as the immunogens. The vaccines elicited strong antibody responses in hamsters and nonhuman primates and protected them from lethal challenge. Since all recent outbreaks in humans were caused by the Bangladesh genotype, we developed new vectors that express the Nipah virus Bangladesh F or G. Those new vectors provided complete protection against disease in hamsters and nonhuman primates. In nonhuman primates protection was achieved against homologous and heterologous challenge. (de Wit et al., manuscript in preparation)